Selective growth media

ABSTRACT

Disclosed is a method of selectively inhibiting growth of non-target cells in a mixed population of target and non-target cells, the method comprising the steps of: (a) contacting the mixed population with a selective agent which comprises a carrier moiety linked by a scissile linkage to a toxic moiety; wherein the selective agent is able to enter non-target cells in which the scissile linkage is cleaved, releasing the toxic moiety to exert a toxic effect on the non-target cells causing inhibition of the growth of the non-target cells, whereas the selective agent is unable to enter target cells and/or the scissile linkage is not cleaved in target cells and/or toxic moiety, if released from the selective agent, does not exert a toxic effect on the target cell; and (b) culturing the cells in conditions which allow for growth of non-inhibited cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority toU.S. Ser. No. 12/762,765 filed on Apr. 19, 2010, which is continuationof and claims priority to U.S. Ser. No. 10/380,330 filed on Sep. 12,2003, now U.S. Pat. No. 7,704,706 B2, which is a National Phase PatentApplication of PCT/GB01/04124 filed on Sep. 14, 2001, which claimsbenefit of Great Britain Patent Application No. 0022556.5 filed on Sep.14, 2000, each of said applications being hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to a method of selectively inhibiting the growthof certain cells in a mixed population, a selective medium for use inthe method, and a kit for performing the method.

BACKGROUND OF THE INVENTION

Many selective agents are known which, when incorporated into biologicalgrowth media, allow for the preferential growth (i.e. selection) ofparticular organisms, especially particular bacteria. It is well-known,for example when performing a bacterial transformation, to incorporatean antibiotic resistance gene on the transforming DNA, and subsequentlyexposing the mixed population of transformed and untransformed cells tothe relevant antibiotic, thereby inhibiting the growth of untransformedcells and selecting for transformed cells.

Equally, it is known to use various dye substances or salts to selectfor a particular organism (e.g. a pathogen) in a mixed population ofbacteria present in a sample obtained from a human or animal subject, asan aid to diagnosis of infectious diseases. However, these selectiveagents are known to inhibit the growth of healthy cells (Vassiliadis etal, 1974 J. Appl. Bacteriol. 37, 411-418) and to restrict the recoveryof injured cells (Kang & Siragusa 1999 Appl. and Env. Microbiol. 65,5334-5337). This is a severe disadvantage because, in many practicalapplications, it is desired to recover organisms which are injured or“stressed” (e.g. when attempting to recover pathogens from food samples)due to exposure to sub-optimal conditions (of temperature, pH, or thelike).

Allen et al. (1978 Nature 272, 56-58) disclosed that phosphonopeptidespossessed antibacterial properties. In particular, the compoundL-alanyl-L-1-aminoethylphosphonic acid (called “alaphosphin”) was shownto be a reasonably potent anti-bacterial agent which was believed tocause inhibition of peptidoglycan synthesis. Alaphosphin consists of theL stereoisomer of alanine, coupled to L-1-aminoethylphosphonic acid(AEP), the —COOH group of the alanine and the amino group of AEPcondensing to form a peptide bond. These original findings were furtherdeveloped by Atherton et al. (1979 Antimicrob. Agents and Chemother. 15,677-683) and by Allen et al. (1979 Antimicrob. Agents and Chemother. 16,306-313). However, alaphosphin was never widely adopted as anantibiotic, and was not proposed for use as a selective agent. Inparticular, antibiotics are generally intended to be “broad spectrum”,so as to kill a wide range of bacteria, which renders their use asselective agents in diagnostic microbiology unlikely.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method of selectivelyinhibiting the growth of non-target cells in a mixed population oftarget and non-target cells, the method comprising the steps of: (a)contacting the mixed population with a selective agent which comprises acarrier moiety linked by a scissile linkage to a toxic moiety; whereinthe selective agent is able to enter non-target cells in which thescissile linkage is cleaved, releasing the toxic moiety to exert a toxiceffect on the non-target cells causing inhibition of the growth of thenon-target cells, whereas the selective agent is unable to enter targetcells and/or the scissile linkage is not cleaved in target cells and/orthe toxic moiety, if released from the selective agent, does not exert atoxic effect on the target cell; and (b) culturing the cells inconditions which allow for growth of non-inhibited cells.

The cells may be eukaryotic cells (e.g. mammalian cells, fungal cells oryeast cells) but more typically will be bacterial cells. In particular,the target and non-target cells will normally both comprise bacteria,and advantageously the target cell may be a Gram negative organism (e.g.Salmonella spp.) and the non-target cells will also comprise Gramnegative organisms (e.g. E. coli).

The target cells will typically be those of an organism whose presenceit is desired to detect among the mixed population. For example thetarget cells may be a particular pathogenic species or genus, whilst thenon-target cells (which are not of interest) may be cells representativeof the normal gut or skin flora of a subject, from whom a samplecontaining the mixed population has been obtained. Alternatively, thesample may be, for example, a sample of a foodstuff or drink for humanor animal consumption. Typically the non-target cells will be present ingreater numbers than the target cells, hence it will be desirableselectively to inhibit the growth of the non-target cells so as tofacilitate detection of the target cells, which would otherwise tend tobe outgrown and so masked by the non-target cells. This is of particularimportance during pre-enrichment when it is possible that the targetcell may be injured or stressed and undergoes an extended lag-phase asthe cell repairs any injury suffered during the manufacture orpreparation of food-stuffs. A proportion of the total population ofcompetitors will not suffer any injury and will not enter a lag-phasewhen inoculated into the pre-enrichment broth. These can grow quicklyand, through a mechanism known as the Jameson Effect (Jameson 1962, J.Hygiene Cambridge 60, 193-207), can prevent the target cell from growingbefore it has left the lag-phase and begun to multiply. This canseverely limit the likelihood of detecting the target cell on subsequentsub-culture.

It will be apparent from the foregoing that there may be more than onebasis for the selectivity of the selective agent, one or more of whichmay operate for a particular selective agent/mixed populationcombination. One basis of selectivity (which may be employed inisolation or, more preferably, in conjunction with another basis ofselectivity) is that of selective uptake by non-target cells, such thatthe selective agent accumulates in non-target cells but does notaccumulate at toxic concentration in target cells. Convenientlyselective uptake may be achieved by making use of uptake mechanisms(especially uptake enzymes such as permeases) operable in the non-targetcell but not present in the target cell. In one convenient embodiment,the selective agent enters non-target cells by means of a dipeptide,tri-peptide or oligopeptide permease. Thus, the selective agent maydesirably comprise a carrier moiety which is efficiently processed by anuptake mechanism in non-target cells.

Another basis of selectivity (which may be employed in isolation or incombination with a different basis) comprises use of a selective agenthaving a scissile linkage which is cleavable by non-target cells but isnot cleavable by target cells. Conveniently, cleavage of the scissilelinkage is effected by an enzyme or combination of enzymes expressed bythe non-target cells but not by the target cells.

In certain embodiments the scissile linkage desirably comprises apeptide bond, cleavable by a peptidase (preferably an aminopeptidase)expressed by non-target cells but not by target cells. Typically thepeptide bond will be formed by the α-COOH group of an amino acid oramino acid analogue, but it is possible that the peptide bond may beformed by a β-COOH group (e.g. aspartic acid) or δ-COOH-group (e.g.glutamic acid).

Advantageously the selective agent comprises one or more amino acidresidues (including unusual amino acids such as hydroxyproline andpyroglutamic acid) or amino acid residue analogues. A number of aminoacid residue analogues suitable for inclusion in the selective agent aredescribed by Allen et al. or Atherton et al., cited above.

Table 1 identifies a number of enzymes which are present in somebacteria, but not in others, and which might therefore be suitable forcleaving a toxic moiety from a selective agent and allowing theselective agent to inhibit the growth of non-target cells over targetcells. In general amino-peptidases are preferred to glycosidases, andpreferred selective agents therefore comprise L-amino acid residues oranalogues thereof. Particularly preferred (especially in the context ofselective media for the growth of Salmonella spp.) are selective agentscleavable by pyrrolidonylarylamidases.

TABLE I Enzyme Classification Aminopeptidases Number Substrate prolylaminopeptidase 3.4.11.5 L-prolyl-AEP leucyl aminopeptidase  3.4.11.10L-leucyl-AEP pyrrolidonylarylamidase 3.4.19.3 L-pyrrolidonyl-AEPGlycosidases α-galactosidase 3.2.1.22 AEP-α-galactoside β-galactosidase3.2.1.23 AEP-β-galactoside β-glucuronidase 3.3.1.31 AEP-β-glucuronide6-phospho-β-galactosidase 3.2.1.85 AEP-β-galactosides6-phospho-β-glucosidase 3.2.1.86 AEP-β-glucosides

It will generally be preferred that when cleaved from the carriermoiety, the toxic moiety will tend to remain within the non-target cellor, after lysis of the non-target cell, remain covalently associatedwith lysed remnants of the non-target cell. In this way, should the onlybasis for selectivity between non-target and target cells be therespective ability or inability to cleave the scissile linkage betweenthe carrier moiety and the toxic moiety, the toxic moiety (once cleavedfrom the carrier moiety inside a non-target cell) will not be releasedinto the extracellular medium and so will not exert any toxic effect onthe target cells. Alternatively, if released from the non-target cell(or used remnants thereof), the toxic moiety should preferably be highlycharged, which tends to prevent passive entry of the free toxic moietyacross the cell membrane of target cell.

Accordingly, in preferred embodiments the toxic moiety should have ahigh charge density (i.e. be highly charged) at the pH and under theconditions in which the selective agent is contacted with the mixedpopulation. This also prevents the toxic moiety from migrating throughthe lipid bilayer of the cell envelope of the non-target cell and intothe extra-cellular environment.

For present purposes a toxic moiety may be considered as having a highcharge density if it has a negative log octanol/water partitioncoefficient, as determined by the method of Meylan & Howard (1995Journal of Pharmaceutical Science 84, 83-92).

By way of explanation, the partition coefficient (logP) of a solute isdetermined most frequently using an octanol and water mixture. Theconcentration of the solute is measured in both phases and expressed asa number according the equation: logP=log [(x)_(octanol)/(x)_(water)](e.g. Leo et al 1971 Chemical Reviews 71, 526-616, Meylan and Howard1995, cited above).

Those compounds that have a positive LogP are more soluble in lipidsthan in water, while those with a negative LogP are more soluble inwater than in lipids.

The majority of biocides have positive LogP, and this is necessaryproperty of membrane active compounds that enable them to disrupt thefunctions of the cytoplasmic membrane of the cell. By contrast, thosecompounds with negative LogP are not membrane active and are preventedfrom entering the cell due to the charge associated with the moleculethat prevents them crossing the cytoplasmic membrane to their site ofaction in the cytoplasm.

Table 2 below shows the log octanol/water partition coefficient forvarious toxic moieties: those having a negative value are especiallyadvantageous for incorporation in a selective agent for use inaccordance with the present invention.

Toxic Moiety Log Partition Coefficient 2-phenylethanol 1.36^(a) Phenol0.88^(a) Dowicide 9 4.48^(b) (4-chloro-2-cyclopentylphenol)4-hydroxybenzoic acid 2.23^(a) 4-methylphenol 1.98^(a) 4-bromophenol2.59^(a) 2,4-dicholorophenol 2.80^(b) 4-chloro-2-nitrophenol 2.55^(b)2-nitrophenol 2.00^(a) ethyl-4-hydroxybenzoic acid 2.45^(b) Thiophenol2.52^(a) Aniline 0.94^(a) 2-mercaptopyridine 1.50^(b)4-[N-(mercaptoethyl)] aminipyridine- 0.63 2,6-dicarboxylic acid8-hydroxyquinoline 1.75^(a) 8-hydroxyquinoline-5-sulphonic acid−1.50^(b) L-l-aminoethylphosphonic acid −1.75^(b) Sulfacetamide−0.60^(b) Sulfanilamide −0.78^(a) Sulfanillic acid −2.08^(b)N³-4-methoxyfumaroyl-L-2,3- −2.90^(b) diaminopropionic acidMetronidazole −0.15^(b) Sulfamethoxazole −0.20^(b) Aminoxy-L-alanine−0.901^(b) Hydrazino-L-alanine −0.908^(b) β-chloro-L-alanine −3.011^(b)^(a)Leo et al (1971); ^(b)calculated by the method in Meylan & Howard(1995).

Preferred toxic moieties include 1-aminoalkyl compounds (especiallyacids and salts) especially amino lower alkyl compounds (i.e. thosecomprising a C₁₋₄ alkyl group, preferably an ethyl group), especiallyacids and salts. Preferred toxic moieties include aminoalkyl phosphonicacids/salts such as 1-aminoethyl phosphonic acid (AEP) and aminoalkylsulphonic acids/salts, such as 1-aminoethyl sulphonic acid (AES). Theseaminoalkyl compounds possess a degree of structural similarity withamino acids, and may therefore be considered, for present purposes, asamino acid analogues, and when incorporated into a selective agent(preferably by a peptide bond) may be considered as amino acid residueanalogues. Indeed, highly charged amino acid analogues may generally beuseful as toxic moieties in the method of the invention. Other exampleswhich may be useful are glutamine analogues, especially glutamineanalogues which inhibit the enzyme glucosamine-6-phosphate synthase.Suitable analogues include, for example,N³-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (abbreviated as FMDP)and related compounds disclosed in GB 2282602. As yet another example,the toxic moiety may be any molecule useful as an inhibitor of L-alanylracemase enzyme, including without limitation, aminoxy-L-alanine,hydrazine-L-alanine, and β-chloro-L-alanine.

Conveniently the 1-amino group of the preferred aminoalkyl acids/saltsmay be joined to a carboxyl group of a carrier moiety to form a peptidebond scissile linkage, cleavable by a peptidase (advantageously anaminopeptidase).

As an alternative the selective agent may comprise a glycoside,especially an N-glycoside, in which the carrier moiety is an N-sugar.For example, suitable selective agents may comprise a 1-aminoalkylcompound as aforementioned, such as AEP or AES or salts thereof,covalently coupled to the N-sugar via the α-amino group.

In certain embodiments the selective agent resembles a di-, tri- oroligopeptide. In one preferred embodiment, the selective agent resemblesa dipeptide, with the carrier moiety comprising an amino acid residue,and the toxic moiety comprising an amino acid residue analogue.Desirably the one or amino acid residues in the carrier moiety are the Lstereoisomer. Equally, a preferred amino acid residue analogue of thetoxic moiety is an L stereoisomer.

In a preferred embodiment, the carrier moiety comprises an L-alanineresidue which, desirably, is joined via a peptide bond via its COOHgroup to an L-alanine residue analogue (such as AEP or AES). Thus in onepreferred embodiment the selective agent comprises a dipeptideconsisting of an L-alanine residue linked to an L-alanine residueanalogue, the selective agent being an alanyl-alanine analogue. In suchan embodiment, the enzyme L-alanyl aminopeptidase (“LALA”) can cleavethe peptide bond (i.e. the scissile linkage) between the L-alanineresidue and the L-alanine residue analogue, releasing the L-alanineanalogue to exert a toxic effect.

Such embodiments are particularly convenient, as the enzyme LALA is notexpressed by all bacteria, and thus can be used as a basis forselectivity. In addition, L-alanine analogues are effective growthinhibitors, acting as essentially irreversible inhibitors of the enzymesinvolved in peptidoglycan synthesis. The L-alanine analogues becometightly associated with the enzymes, and so tend not to be released intothe extracellular environment in a free, toxic form, even after lysis ofthe non-target cell.

The term ‘analogue’ as used herein, will therefore be understood bythose skilled in the art to refer to a molecule which shares areasonable degree of structural similarity with the parent molecule ofwhich it is an analogue and, in particular, an enzyme which acts on theparent molecule will generally bind to an analogue thereof. However, dueto the differences between the parent molecule and the analogue, theenzyme may not process the two entities in the same way. For example,whilst the parent molecule will be a substrate for the enzyme and willbe released from the enzyme once the enzyme-catalysed reaction has takenplace, the analogue will not necessarily be subject to theenzyme-catalysed reaction undergone by the parent molecule, and so mayremain bound to the enzyme and act as a potent inhibitor thereof.

However, those skilled in the art will appreciate that a large number ofother peptidases (especially aminopeptidases) are expressed byparticular groups of organisms and selective agents for use in theinvention may comprise other amino acid residues or amino acid residueanalogues. Examples of amino acids or analogues suitable for inclusionas the carrier moiety include valine, proline and pyroglutamic acid(pyr). Thus, for example, proline-AEP, proline-AES or Pyr-AEP andPyr-AES represent other suitable selective agents for use in theinvention.

A number of bacteria possess a dipeptide, tripeptide or oligopeptidepermease, which facilitates entry into the cell of dipeptide, tripeptideor oligopeptide selective agents, which allows relatively lowconcentrations of selective agent to be used effectively.

The method of the invention may be applied in any manner of situationswhere it is desired to cause inhibition of part of a mixed population ofcells. Examples include: selective inhibition of coliforms (especiallyE. coli) in mixed populations of bacteria in clinical samples, so as tofacilitate isolation of Campylobacter (which do not possess L-alanylaminopeptidase activity and which will, if present, typically constituteonly a very minor portion of the mixed population); selective inhibitionof Gram negative bacteria (especially coliforms) to facilitate isolationof pathogenic Gram positive organisms (such as Staph aureus which isL-alanyl aminopeptidase-ve): and distinguishing in clinical samplesbetween the presence of Haemophilus influenzae and Haemophilusparainfluenzae, (which distinction has implications for prognosis andtreatment), by culturing samples in suitable conditions with a selectiveagent so as to cause selective inhibition of one of the aforesaidorganisms (H. influenzae has no relevant aminopeptidase activity, whilstH. parainfluenzae does possess a relevant aminopeptidase which willcleave compounds such as alaphosphin).

A further particular application of the present invention, which isespecially preferred, is to selectivity inhibit Citrobacter and othercoliforms in mixed populations of bacteria so as to facilitatedetection, and optionally isolation, of Salmonella spp: e.g. from foodsamples, so as to assist in diagnosis of disease and to identifycontaminated food samples in public health measures. In such anembodiment, it may be desirable to use a selective agent comprising atoxic moiety linked via a peptide bond to pyroglutamic acid, since theinventors have noted that Salmonella spp generally lacked a pyroglutamylpeptidase, whilst such an enzyme is present in most coliforms, so thatthe non-target organisms will cleave the toxic moiety from the selectiveagent.

Conventionally, when testing samples of food and the like for possiblecontamination with Salmonella, it is usual to carry out a two-stepincubation. In the first step about 25 gms of sample is usual diluted1/10 in 225 mls of pre-enrichment broth and incubated for about 16 hoursor overnight. The pre-enrichment broth usually does not contain anyselective agent. This is because Salmonella organisms possibly presentin the sample will frequently be “stressed” (that is are weakened havingbeen exposed to suboptimal conditions of temperature, pH and the like).In such a “stressed” condition the presence of a selective agent at anormally sub-lethal concentration may often cause cell death.

Incubation in the pre-enrichment medium allows any stressed cells torecover. The cultures are then further diluted (e.g. 100 μl into 10 mls)into an enrichment medium which contains a selective agent at aconcentration which allows Salmonella spp. to grow, whilst inhibiting(i.e. preventing any net increase in viable cell numbers) competitorcoliform organisms. In practice, most conventional selective agents alsoinhibit the growth of Salmonella spp, but to a significantly lesserextent than they inhibit competitor organisms.

An advantage of the present invention, in preferred embodiments, is thata selective agent can be used which is substantially non-inhibitory toSalmonella spp, even in a stressed state. Accordingly it is possible toinclude the selective agent in the initial medium and/or to reduce theoverall culture time required for Salmonella spp organisms (if presentin the original sample) to attain the cell density required to give apositive result in any assay for their presence (e.g. ELISA, PCR etc),since their growth is not inhibited. This permits the results to be madeavailable sooner (following receipt of the sample) than has hithertobeen the case.

In preferred embodiments of the invention the selective agent causesinhibition of the non-target cells but is essentially non-inhibitory totarget cells, whether they are in stressed or unstressed condition.

When bacterial cells are placed in a suitable growth medium there is a‘lag phase’ during which the net number of viable bacterial cells doesnot increase, or increases only slowly. After the lag phase, the cultureenters an exponential growth phase in which the mean “generation time”(that is, the mean time taken for a number of cells to proceed fromformation to fission) is at its shortest.

As an illustration of what is considered “essentially non-inhibitory”, aselective agent will normally be considered essentially non-inhibitoryto target cells at a particular concentration if: it causes an increasein the lag phase of less than 25%, preferably less than 20% and morepreferably less than 15% and if it causes an increase in the meangeneration time, during the exponential growth phase, of less than 20%,preferably less than 10%, and more preferably less than 5%.

Those skilled in the art will appreciate that performance of the methodof the first aspect of the invention may allow conclusions to be maderegarding the identity of organisms which are able to grow successfullyin the selective growth conditions. Thus, in some embodiments, theinvention may comprise the further step of identifying target cellorganisms which are able to grow in a culture comprising the selectiveagent. Alternatively, or additionally, the method may comprise the stepof isolating colonies of the target cell organisms which are able togrow in a culture comprising the selective agent. Such methods ofidentification and/or isolation are routine for those skilled in the artand form no part of the present invention.

In a second aspect the invention provides a selective medium forselective inhibition of non-target cells in a mixed population ofnon-target cells and target cells, the medium comprising a selectiveagent, which selective agent comprises a carrier moiety linked via ascissile linkage to a toxic moiety. Preferably the selective agent is asdefined above. The selective medium may be liquid or solid, and maycomprise any of the components which may conventionally be included inmedia, such as peptones, yeast extract, agar (or other solidifyingagent), salts, buffers, indicator dyes and the like.

In a third aspect, the invention provides a kit for causing selectiveinhibition of non-target cells in a mixed population comprisingnon-target cells and target cells, the kit comprising a selective agentas defined above and instructions for use in accordance with the methodof the first aspect of the invention.

In preferred embodiments, the kit will comprise a medium in accordancewith the second aspect of the invention defined above or, as analternative, ingredients for preparing a selective medium in accordancewith the invention.

The various aspects of the invention will now be further described byway of illustrative example and with reference to the accompanyingdrawings in which:

FIG. 1 a is a representation of the chemical structure a preferred toxicmoiety (AEP) for use in the method of the invention;

FIG. 1 b is a representation of the chemical structure of a preferredselective agent, comprising the toxic moiety illustrated in FIG. 1 a;

FIG. 1 c illustrates cleavage of the selective agent shown in FIG. 1 b,by an aminopeptidase, to release the toxic moiety shown in FIG. 1 a;

FIGS. 2-12 are bar charts showing the amount of growth of variousorganisms, in pure or mixed culture, in the presence of AEP oralaphosphin;

FIGS. 13, and 16-19 are graphs showing growth of various bacteria (asmeasured by Optical Density) against time (hours) under variousconditions;

FIGS. 14 and 15 are graphs showing growth of various bacteria (asmeasured by Log₁₀ Cfu/ml) against time (hours) under various conditions;

FIG. 20 is a bar chart showing the effect of various media on therecovery of heat-stressed S. typhimurium cultures; and

FIG. 21 shows the structure of a particular selective agent(L-pyroglutamyl-L-1-aminoethylphosphonic acid) suitable for use in themethod of the invention.

FIG. 22 a is a photograph of an incubated DMSal agar plate withoutsulfacetamide-β-D-galactoside seeded with E. faecalis only.

FIG. 22 b is a photograph of an incubated DMSal agar plate withoutsulfacetamide-β-D-galactoside seeded with E. faecalis and streaked withStaphylococcus aureus.

FIG. 22 c is a photograph of an incubated DMSal agar plate containingsulfacetamide-β-D-galactoside (20 μg/ml) seeded with E. faecalis only.

FIG. 22 d is a photograph of an incubated DMSal agar plate containingsulfacetamide-β-D-galactoside (20 μg/ml) seeded with E. faecalis andstreaked with S. aureus.

In FIGS. 1 b and 1 c, R may be inter alia, any of the side chains of theamino acid residues. In FIG. 1 b, where R is CH₃, the selective agent isalaphosphin, and the released L-amino acid in FIG. 1 c is L-alanine.

EXAMPLES

In the Examples that follow, a large number of bacterial strains arementioned. The letters “OCC” are an abbreviation for ‘Oxoid CultureCollection’, and these organisms were obtained from within theApplicant's own laboratories. However, in most instances, identical (orat least closely equivalent) organisms are obtainable from publiclyavailable collections such as the National Collection of Type Cultures(NCTC, Central Public Health Laboratory, Colindale, London) or theAmerican Type Culture Collection (ATCC, Manassas, Va., USA) orelsewhere, as shown in Table 3 below. In any event, these organisms aremerely representative samples and other strains, typical of the speciesin question, could equally be used for the purposes of illustrating theinvention.

TABLE 3 Oxoid Culture Collection Strain Number (OCC) NCTC ATCC OtherCitrobacter freundii PHLS Poole 93703 Citrobacter Freundii 261Enterobacter aerogenes 720 10006 13048 Enterobacter cloacae 760 1000513047 Escherichia coli 122 Escherichia coli 1872 12900 O 157:H7 VT-veEscherichia coli 10090 Escherichia coli 402 9001 11775 Escherichia coli2129 Bacillus subtilis 214 6633 Enterococcus faecalis 640 29212Klebsiella pneumonia CMCC 3077 Klebsiella pneumonia 411 29665Staphylococcus aureus 102 Salmonella enteritidis 723 25928 Salmonellaindiana 2412 11304 Salmonella typhimurium 722 12023 14028 Salmonellavirchow 703 5742 Salmonella typhimurium 1792 CMCC 3073 SalmonellaWorthington 634

Example 1

A number of experiments were conducted to demonstrate inhibition ofgrowth of certain bacteria and selective inhibition of bacteria in mixedpopulations of cells. In these experiments, described in Examples 1-5,bacteria were incubated on nutrient agar (CM3) supplemented withalaphosphin or AEP (both obtained from Fluka). The experiments wereconducted as follows: nutrient agar medium was autoclaved and, oncooling (but prior to setting) filter-sterilised alaphosphin or AEP wereincorporated at final concentrations of 1 mM, 2 mM, 5 mM and 10 mM, andthe medium used to pour plates. Control plates were prepared usingnutrient agar without alaphosphin or AEP. A single colony of theorganism under test was inoculated in 10 ml of nutrient broth andincubated (without agitation) at 37° C. for 4 hours. The culture wasthen diluted 1:1000 (in nutrient broth) and the resulting dilution usedto inoculate the prepared plates using a variant of the “econometric”method (Mossel et al, 1983 J. Appl. Bacteriol. 54, 313-327) with 4streaks from a 1 μl inoculating loop. (Where mixed populations whereprepared, as in Examples 6-8, 11 samples were taken from the respective1:1000 dilutions of the separate cultures, the 1 μl samples mixedtogether and then used to inoculate the plates as described previously).

In example 1 the sensitivity of Bacillus subtilis strain OCC 214 toalaphosphin or AEP was investigated. The results are shown in FIG. 2,which is a bar chart showing the number of streaks demonstratingbacterial growth at 0-10 mM concentrations of alaphosphin (blankcolumns) or AEP (shaded columns). The results indicate that B. subtilisOCC214 is inhibited by AEP at a concentration of 5 mM or more, but iscompletely insensitive to alaphosphin and/or cannot hydrolysealaphosphin to release AEP. The organism is known to be LALA-ve, so theobservations agree with the known characteristics of the organism.

Example 2

Example 1 was repeated, using Salmonella typhimurium OCC 1870 as thetest organism. The results are shown in FIG. 3, which uses the same keyas FIG. 2. S. typhimurium OCC 1870 was found to be resistant to AEP atconcentrations as high as 10 mM. However, the organism is known to beLALA+, and alaphosphin was found to be completely inhibitory atconcentrations of 5 mM or more, indicating that alaphosphin is taken upby the cell and hydrolysed, but that AEP itself cannot enter the cell.

Example 3

Example 1 was repeated using Enterococcus faecalis OCC 640 as the testorganism. The results are shown in FIG. 4. The organism was found to becompletely inhibited by AEP at a concentration of 5 mM or more,indicating that the organism expresses an uptake polypeptide (possiblyan L-alanine permease) which accepts and transports AEP. The organism isLALA+ and is completely inhibited by alaphosphin at a concentration of 2mM or more.

Example 4

Example 1 was repeated using Klebsiella pneumoniae CMCC 3077 as the testorganism. The results are shown in FIG. 5, and are qualitatively similarto those obtained in example 2. K. pneumoniae CMCC 3077 is LALA+ve andis completely inhibited by alaphosphin at 2 mM or more, but isinsensitive to AEP at concentrations up to 10 mM.

Example 5

Example 1 was repeated using Staphylococcus aureus OCC 102 as the testorganism. The results are shown in FIG. 6. S. aureus OCC 102 wascompletely insensitive to AEP at all concentrations tested, but wascompletely inhibited by alaphosphin at 10 mM. This indicates that theorganism can take up alaphosphin and hydrolyse it, but less efficientlythan the organisms tested in Examples 2-4.

Example 6

Example 1 was repeated, this time using a mixed culture of K. pneumoniaeCMCC 3077 and S. aureus OCC 102. The results for growth in the presenceof AEP or alaphosphin are shown in FIGS. 7 and 8 respectively: emptycolumns represent growth of S. aureus, the shaded columns representgrowth of K. pneumoniae. As predicted from Examples 4 and 5, neitherorganism was inhibited by AEP in mixed culture. In the presence ofalaphosphin, K. pneumoniae growth was completely inhibited above 1 mM,whilst S. aureus was inhibited only by 10 mM alaphosphin. Accordingly,there is a considerable alaphosphin concentration range over which S.aureus will readily grow whilst K. pneumoniae will be inhibited. Even ifK. pneumoniae cells lyse due to the toxic effect of AEP releasedintracellularly, the AEP is not toxic for S. aureus at theconcentrations involved.

Example 7

Example 6 was repeated, this time using a mixed culture of S. aureus OCC102 and E. faecalis OCC 604, in the presence of AEP or alaphosphin. Theresults are shown in FIGS. 9 and 10 respectively. The empty columnsrepresent growth of S. aureus, the shaded columns represent growth of E.faecalis. Alaphosphin at a concentration of 2 mM or more caused completeinhibition of E. faecalis, whilst S. aureus was inhibited only at aconcentration of 10 mM or more. Thus alaphosphin could be used, whenincorporated at an appropriate concentration in a medium, to inhibitgrowth of organisms such as Enterococci in samples whilst allowingStaphylococci to grow and be detected.

Example 8

Example 6 was repeated, this time using a mixed culture of B. subtilisOCC 214 and K. pneumoniae CMCC 3077, in the presence of AEP oralaphosphin. The results are shown in FIGS. 11 and 12 respectively. Theempty columns show the growth of B. subtilis, the shaded columns denotegrowth of K. pneumoniae. FIG. 11 shows that K. pneumoniae is insensitiveto AEP at all the concentrations tested, whilst B. subtilis exhibitedsome sensitivity above 5 mM. FIG. 12 shows that K. pneumoniae wascompletely inhibited by alaphosphin at a concentration of 2 mM or more,whilst B. subtilis was insensitive to alaphosphin at this concentrationrange. The results indicate that hydrolysis of alaphosphin by K.pneumoniae does not yield sufficient free AEP to cause any inhibition ofB. subtilis.

The above examples all relate to experiments conducted using nutrientagar, which contained peptone at 8 gms/liter. Peptone containsoligopeptides which may affect expression of oligopeptide permeaseand/or peptide genes. Accordingly, where the selective agent is a di-,tri- or oligopeptide, and/or the scissile linkage comprises a peptide,the type and/or concentration of peptone in the selective medium mayaffect the concentration of selective agent required in the medium toobtain the optimum degree of selectivity.

Example 9

Other compounds which should prove useful as a selective agent in amethod in accordance with the invention are those in which the carriermoiety comprises pyroglutamic acid (Pyr), especially L-pyr. Convenientlythe toxic moiety will comprise AEP. Such a selective agent should beable selectively to inhibit the growth of organisms expressing a“Pyrase” (i.e. Pyr⁺ organisms), whilst allowing Pyr^(−ve) organisms togrow. Many coliforms are Pyr⁺ (e.g. Citrobacter spp., Klebsiella spp.,Serratia spp. and most Enterobacter spp.). Thus, for example, L-pyr-AEPmight be a useful selective agent in liquid media to facilitate theselective pre-enrichment step during isolation of Salmonella spp. fromclinical or environmental samples, and subsequently in solid media forthe selective isolation step.

In another embodiment L-pyr-AEP might be used for selective enrichmentfor Pyr^(−ve) Listeria spp, whilst inhibiting Pyr⁺ coliforms,enterococci and many Bacillus spp.

A number of experiments were performed in order to illustrate theseembodiments of the invention.

Example 9A

Data and explanatory text for inclusion in Example 9 (Pyr-AEP).

This example demonstrates the inhibition, by L-pyroglutamyl-L-aminoethylphosphonate (Pyr-AEP), of organisms genotypically similar to Salmonellabut not Salmonella itself. Thus, Pyr-AEP was dissolved in deionisedwater (14.1 mg/ml), filter sterilised, and an amount of the solutionadded to autoclaved Lab-Lemco broth (20 g/l; pH 6.0) to give a finalconcentration of 141 μg/ml. Volumes (300 μl) of this solution were thenpipetted into the wells of Bioscreen microtitre plates and 30 μlquantities of 1 in 10,000 dilutions of overnight cultures of Citrobacterfreundii, Enterobacter aerogenes, Enterobacter cloacae, Salmonellatyphimurium and Salmonella worthington added (note that this furtherdilution reduced the concentration of Pyr-AEP to 128 μg/ml). Plates werethen covered, incubated at 37° C., and the opacity of the organismsuspensions measured using a Bioscreen instrument (a semi-automatedmicrobiological growth analyser, available from Thermolabsystems,Ashford, Middlesex, U.K.). Growth of the Salmonella strains, which donot contain L-pyroglutamyl hydrolase (also referred to as apyrrolidonylarylamidase, E.C. 3.4.19.3), was not inhibited by Pyr-AEPwhereas the other strains tested, which do contain the enzyme, wereinhibited.

The results are shown in FIG. 13, which is a graph of optical densityagainst time (in hours). The optical density readings were taken in“wide band” (wb) mode, i.e. using white light without any filter.

Example 9B

In addition to the pure culture work described above, mixed cultureexperiments were performed.

L-Pyroglutamyl-L-aminoethylphosphonate was added to Oxoid Nutrient BrothNo. 2 (final concentration=128 μg/ml) contained in a universal tube in awater bath at 37° C. Citrobacter freundii OCC 370 and Salmonellatyphimurium OCC 626 were grown overnight in Nutrient Broth No. 2 at 37°C., diluted 1 in 1000 in Maximal Recovery Diluent and amounts added tothe Nutrient Broth to give final concentrations of about 10⁴ cfu/ml ofeach organism. At intervals samples were taken and spread on XLD and thenumber of red colonies with black centres enumerated as Salmonella andyellow ones as Citrobacter.

The results are shown in FIG. 14, which is a graph of Log₁₀ viable count(cfu/ml) against time (in hours). The plots for C. freundii are shown bylozenge symbols, whist those for S. typhimurium are shown by squares.Filled symbols and solid lines show results in the presence of Pyr-AEP,empty symbols and dotted lines show results in the absence of Pyr-AEP.

The graph clearly shows that Pyr-AEP has no significant effect on thegrowth of Salmonella, but is completely inhibitory for Citrobacter.

Example 10

Growth curve data on the activity of alaphosphin towards Klebsiella spp.was generated. Particular attention was paid to measuring the kineticsof inhibition in broth culture. The media used in this work wereBuffered Peptone Water, Nutrient Broth and Tryptone Soya Agar. The mediawere prepared according to the manufacturer's instructions.

The selective agents, AEP (Cat. No. 06655) and alaphosphin (Cat. No.05260), were supplied by Fluka. They were dissolved in water at 0.5Mconcentration, filter-sterilised, and 100 μl added to 10 ml of NutrientBroth to give a final concentration in the test medium of 5 mM of AEP oralaphosphin respectively.

Klebsiella pneumnoniae CMCC3077 was chosen, as it is a food isolate andwork had been carried out previously on this organism using alaphosphinand AEP (see preceding examples). The organism was inoculated intoNutrient Broth and incubated overnight at 37° C. The culture was dilutedand inoculated into the test media so that it had a final concentrationof between 1×10⁵ cfu/ml and 1×10⁶ cfu/ml. The test media were incubatedat 37° C., and sampled at hourly intervals. The cultures were seriallydiluted in Buffered Peptone Water and inoculated onto Tryptone SoyaAgar. These plates were incubated overnight at 37° C. and the totalviable count made the next day.

Five mM AEP in Nutrient Broth had little effect on the growth ofKlebsiella pneumoniae CMCC3077 when compared with the Nutrient Brothcontrol. In contrast alaphosphin was an effective selective agent forthe inhibition of Klebsiella pneumoniae CMCC3077 and caused a two tothree log reduction in the viable count in Nutrient Broth within a 4hour period.

Typical results are shown in FIG. 15 which is a graph of total viablecount (log₁₀ cfu/ml) against time (hours). The empty lozenge symbolshows the results in Nutrient Broth control media, the filled squaresshow the results of test media containing 5 mM AEP, and the filledtriangles show the results for test media containing 5 mM alaphosphin.

The concentration of alaphosphin used in this medium was not optimised.The example illustrates that AEP peptides can significantly affect someof the organisms that compete with Salmonella spp. for nutrients inculture media, and that if the substrate is hydrolysed and AEP releasedin to the medium, it has minimal influence on growth rate.

Example 11

Experiments were performed to demonstrate the inhibition, by alaphosphin(Ala-AEP), of strains of Escherichia coli but not Salmonella. Thus,Ala-AEP was dissolved in deionised water (7.0 mg/ml), filter sterilised,and an amount of the solution added to autoclaved Lab-Lemco broth (20g/l; pH 5.7) to give a concentration of 70 μg/ml. Volumes (300 μl) ofthis solution were then pipetted into the wells of Bioscreen microtitreplates and 30 μl quantities of 1 in 10,000 dilutions of overnightcultures added (reducing the final alaphosphin concentration to 64μg/m). Plates were then covered, incubated at 37° C., and the opacity ofthe organism suspensions measured using a Bioscreen instrument. Growthof the Salmonella strains was unaffected whereas the E. coli strainswere all inhibited. In this instance, Salmonella are thought to be ableto take up and hydrolyse the inhibigen but at much reduced rates incomparison with E. coli, such that the growth of the Salmonellae was notsignificantly inhibited.

Typical results are shown in FIG. 16, which is a graph of OpticalDensity against time (hours). Growth of S. indiana OCC 2412 and S.worthington OCC 634 is denoted by crosses and circles respectively. Fourstrains of E. coli tested did not grow at all in the experimentalconditions and the plots for these organisms therefore appear as a solidflat line.

Example 12

Conventional selective agents effectively promote the growth of targetmicroorganisms, e.g. Salmonella by inhibiting the growth of non-targetbacteria through the use of antibiotics, chemicals, and dyes, increasedincubation temperature and reduced pH. Because of the broad andrelatively non-specific nature of these treatments the growth of thetarget microorganism is often negatively affected. That is, theselective agent is not truly specific and causes significant inhibitionof the growth of target organisms. Thus, in conventional methods,incubation times of 16-24 hours may be required in order to attain therequired concentration of target cells for detection.

In contrast, the present invention allows for the possibility ofmaintaining the optimum growth rate of the target organism, which ishighly advantageous in the development of rapid diagnostic tests.

The following is an example to illustrate the beneficial lower toxicproperties of the new selective agents towards the target microorganism,in this case Salmonella. Two selective agents, cefsulodin andnovobiocin, as used conventionally in Salmonella isolation from foods(Humphrey and Whitehead, 1992 British Poultry Science 33, 761-768) werecompared with alaphosphin. All were dissolved in deionised water, filtersterilised, and an amount of the solution added to either autoclavedLab-Lemco broth+phosphate buffer (20 g/l; pH 6.0; ala-AEP) or BufferedPeptone Water (pH 6.8; cefsulodin and novobiocin) to give finalconcentrations of 66, 16.5 and 11 μg/ml respectively. [The different pHvalues reflect the different pH optima of the selective agents (ala-AEPwas also subsequently used in BPW at pH 6.8 and similar resultsobtained)]. Volumes (300 μl) were then pipetted into the wells ofBioscreen microtitre plates and 30 μl quantities of 1 in 10,000dilutions of overnight cultures of E. coli OCC 2129 and Salmonellatyphimurium OCC 722, added. Plates were then covered, incubated at 37°C., and the opacity of the organism suspensions measured using aBioscreen instrument.

Typical results are shown in FIGS. 17-19, which are graphs of opticaldensity against time for S. typhimurium (circles) and E. coli (squares)in the presence (empty symbols) or absence (filled symbols) of 15 μg/mlcefsulodin (FIG. 17), 10 μg/ml novobiocin (FIG. 18) or 60 μg/mlalaphosphin (FIG. 19). It is apparent from these results that bothcefsulodin and novobiocin significantly inhibit the growth of S.typhimurium. Thus, in FIGS. 17 and 18 growth curves of S. typhimurium inthe presence or absence of these two agents are substantially divergentand, in each case, at 12 hours E. coli substantially outgrows S.typhimurium in the presence of the selective agent. In contrast, in FIG.19 it is apparent that there is no significant difference in the growthof S. typhimurium in the presence or absence of alaphosphin at 60 μg/ml.Moreover, growth of E. coli is completely inhibited, allowing S.typhimurium easily to outgrow its competitor.

Example 13

Medium composition was found to affect the inhibition of growth oforganisms by pyroglutamyl-1-aminoethylphosphonic acid. In general, mediacontaining a higher concentration of short chain peptides, for examplehydrolysed casein, reduced the effectiveness of the inhibitor. Thus,Pyr-AEP was dissolved in deionised water (51.2 mg/ml), filtersterilised, and amounts of the solution added to autoclavedBacteriological Peptone, Casein Hydrolysate, Lab-Lemco broth andProteose Peptone (20 g/l; pH 7.3) to give a dilution series of 1,477,739, 369, 185 μg/ml. Volumes (30 W) of these solutions were thenpipetted into the wells of Bioscreen microtitre plates and 30 μlquantities of 1 in 10,000 dilutions of overnight cultures of Citrobacterfreundii OCC 261, Enterobacter aerogenes OCC 720, Salmonella enteritidisOCC 723 and Salmonella virchow OCC 703 added. Plates were then covered,incubated at 37° C., and the opacity of the organism suspensions,measured using a Bioscreen instrument. From the resulting growth curves,MIC values were generated as shown in Table 4 below.

TABLE 4 Minimum inhibitory concentrations of L-Pyroglutamyl-1-Aminoethylphosphonic acid-aep (μg/ml) in various media Medium CaseinBacteriological Proteose Strain Hydrolysate Peptone Peptone Lab-LemcoSalmonella >1343 671 1343 1343 enteritidis OCC723 Salmonella >1343 6711343 1343 virchow OCC703 Citrobacter >1343 168 1343 168 freundii OCC261Enterobacter >1343 671 >1343 336 aerogenes OCC720

Example 14

In view of the importance of being able to recover stressed cells,experiments were conducted to investigate if free AEP had any toxiceffect on stressed cells.

Salmonella typhimurium (OCC 1792) was heat stressed at 51.4° C. for 25minutes according to the protocol of Stephens et al. (1997 J. Appl.Micro. 8, 445-455). Cells were diluted in 7 different resuscitationmedia: yeast extract broth with a high reactive oxygen species contentthat is inhibitory to stressed cells, SPRINT enrichment brothsupplemented with Oxyrase that is an optimised resuscitation medium, BPWas the control that has typical resuscitation properties, BPWsupplemented with NaCl that is inhibitory to stressed cells, and BPWsupplemented with 3 different levels of AEP. Resuscitation wasquantified using a microtitre MPN method.

Typical results are shown in FIG. 20 which is a bar chart showing thedifference in growth (Log cfu/ml) of the stressed cells in various mediarelative to growth in BPW alone. The chart shows that the addition ofAEP did not significantly affect the resuscitation of heat-stressedSalmonella (the variability of the MPN technique being +/−0.25 log).

For the experiments described in Examples 15-17, bacteria were incubatedon defined medium optimized for growth of Salmonella spp. (DMSal)previously disclosed in international patent application WO 2005/067939to Bovill et al. The formulation for DMSal is provided in Table 5.Experiments were conducted as described in Example 1 above except thatother selective reagents (toxic moiety coupled to a carrier moiety) asspecifically noted within each of the following examples, were usedinstead of alaphosphin.

TABLE 5 Material DMSal L-arginine 0.1 L-aspartic acid 0.1 L-cysteine 0.1Glycine 0.1 L-histidine 0.1 L-isoleucine 0.1 L-leucine 0.1 L-lysine HCL0.1 L-methionine 0.1 L-phenylalanine 0.1 L-proline 0.1 L-serine 0.1L-threonine 0.1 L-tryptophan 0.1 L-tyrosine 0.1 L-valine 0.1 Adenine0.01 Cytosine 0.01 Guanine 0.01 Uracil 0.01 calcium pantothenate 0.05choline chloride 0.005 myo-inositol 0.009 Nicotinamide 0.005 pyridoxalhydrochloride 0.005 Riboflavin 0.0001 thiamine hydrochloride (aneurine)0.005 ammonium sulphate 1 calcium chloride 0.005 magnesium sulphateheptahydrate 0.1 sodium citrate 0.5 dipotassium hydrogen phosphate 4.72potassium dihydrogen phosphate 3.77 yeast extract 0.1 Glucose 5 sodiumpyruvate 0.5 Total g/L 17.41

Example 15

This example demonstrates the inhibition or growth of microorganisms onDMSal supplemented with tryptophan-AEP (Trp-AEP). As shown in Table 6,Trp-AEP inhibited most strains of Enterobacteriaceae tested. All E. coliand Shigella strains tested had MIC values of 0.5 μg/ml or less andCitrobacter, Hafnia alvei and Cronobacter sakazakii strains had minimuminhibitory concentration (MIC) values of approximately 1 μg/ml. Otherstrains had MIC values of 2 μg/ml or more with several microorganismsappearing to be completely resistant to Tryp-AEP.

TABLE 6 MIC values of tryptophan-AEP for organisms on DMSal agar No. ofMIC Organism Strains (μg/ml) Gram-negative Acinetobacter baumanii 1 >256Aeromonas hydrophilia 1 128 Burkholderia cepacia 1 >256 Campylobactercoli 4 >128 Campylobacter jejuni 4 >128 Citrobacter freundii 4 1-4Enterobacter aerogenes 1 1 Enterobacter cloacae 2 4 Cronobactersakazakii 1 1 Escherichia coli 7 0.25-0.5  Escherichia hermanii 1 0.5Haemophilus influenzae. 5 >128 Hafnia alvei 1 1 Klebsiella aerogenes 1 8Klebsiella pneumoniae 2 2-4 Proteus mirabilis 1 >128 Proteus vulgaris1 >128 Salmonella 16  2-16 Serratia marcescans 1 2 Shigella 3 0.25-0.5 Yersinia enterocolitica 2 4-8 Vibrio 8  256−>256 Gram-positive Bacilluscereus 2  32−>256 Bacillus subtilis 2  16−>256 Group A Streptococcus7 >256 Streptococcus agalactiae 5 128 Group C Streptococcus 7 256 >256Enterococcus faecalis 6   8−>256 Enterococcus faecium 1 >256 Group DStreptococcus 2 16 Group F Streptococcus 2 >256 Group G Streptococcus 8  8−>256 Listeria monocytogenes 4  64−>128 Listeria innocua 1 32Listeria ivanovii 1 >128 Staphylococcus aureus 14 0.5-16  Staphylococcusepidermidis 3  4-16 Staphylococcus haemolyticus 1 2 Staphylococcussaprophyticus. 1 64 Streptoccocus viridans 1 >256 Streptococcuspneumonaie 5 >256

Example 16

Trp-AEP was compared to alaphosphin (Ala-AEP) for selective growth ofSalmonella strains on DMSal agar. A concentration of 1.5 μg/ml ofAla-AEP is needed to inhibit the growth of E. coli strains. It wasobserved that Trp-AEP added to DMSal at a concentration of 0.25 μg/mlproduced equivalent results to Ala-AEP at a concentration of 1.5 μg/ml,making the Trp-AEP less expensive to use.

TABLE 7 MIC values of Trp-AEP and Ala-AEP in DMSal MIC of Trp- Ala-AEPOrganism AEP (μg/ml) (1.5 μg/ml) Citrobacter freundii 370 2 GrowthCronobacterr sakazakii 1888 0.125 NG Escherichia coli 122 0.25 NGEscherichia coli 481 0.064 NG Escherichia coli 199 0.25 NG Klebsiellaaerogenes 323 3 Growth Klebsiella pneumoniae 411 2 Growth Klebsiellapneumoniae 758 0.064 NG Serratia marcescens 217 3 Growth Salmonellaenteriditis 723 3 Growth Salmonella typhimurium 722 3 Growth Salmonellaarizonae 1200 2 Growth Salmonella virchow 703 3 Growth

Example 17

Isoleucyl-AEP and sarcosyl-alanyl-AEP were compared to ala-AEP (all inDMSal medium) for the ability to selectively recover two clinicallyimportant pathogenic bacteria: Salmonella and Staphylococcus aureus.Table 8 shows the MIC values determined by the experiments.

For selective growth of Salmonella inhibition of Citrobacter,Enterobacter sakazakii and E. coli is important. As shown in Table 8,the MIC values for these undesired bacteria are lower when usingsarcosyl-alanyl-AEP than when using isoleucyl-AEP or ala-AEP.Sarcosyl-alanyl-AEP used at a concentration of 1.0 μg/ml is sufficientto inhibit the growth of all three undesired bacteria while having noeffect on growth of Salmonella typhimurium. This result is comparable tothe performance of ala-AEP. Alternatively, a concentration of 2.0 μg/mlof isoleucyl-AEP is required to inhibit the growth of Citrobacter,Enterobacter sakazakii and E. coli and this concentration has no effecton the Salmonella typhimurium strain tested.

It was observed that isoleucyl-AEP and sarcosyl-alanyl-AEP allow betterrecovery of S. aureus than does ala-AEP. Further, undesired growth ofEnterococcus faecalis and Enterococcus faecium strains was found to bedifficult to inhibit with ala-AEP at concentrations that would permitgrowth of S. aureus strains. However, the MIC values for Enterococcusfaecalis and Enterococcus faecium in the presence of isoleucyl-AEP, orEnterococcus faecalis in the presence of sarcosyl-alanyl-AEP, aregenerally lower than the MIC values for S. aureus strains and allow theselective growth of S. aureus.

TABLE 8 MIC values (μg/ml) of AEP-eontaining selective agents Organismsala-AEP sarcosyl-alanyl-AEP isoleucyl-AEP Citrobacter freundii 370 1.01.0 2.0 Enterobacter sakazakii 0.075 0.01 2.0 1888 E. coli 607 0.1 0.052.0 Salmonella typhimurium 2.0 2.0 10 722 Enterococcus faecalis 10 8 5501 Enterococcus faecalis 10 8 5 581 Enterococcus faecalis 10 16 5 640Enterococcus faecium >50 >256 5 220 S. aureus 198 5 >100 not tested S.aureus ss aureus 2355 10 >100 >200 S. aureus (MRSA) 934 25 >100 nottested

Example 18

The effectiveness of sulfacetamide-β-D-galactoside as a selective agentfor recovery of S. aureus in the presence of E. faecalis was tested.Sulfacetamide-β-D-galactoside (20 μg/ml) and horse serum (50 ml/L) wasadded to DMSal agar to make the test plates; DMSal agar withoutsulfacetamide-β-D-galactoside served as the control. All plates (withand without sulfacetamide-β-D-galactoside) were seeded with E. faecalis640 and half of the total number of plates (with and withoutsulfacetamide-β-D-galactoside) were streaked with S. aureus 106. Allplates were incubated for approximately 24 hours at 37° C.

As FIGS. 22 a and 22 b show, all plates withoutsulfacetamide-β-D-galactoside supported growth of E. faecalis. Whensulfacetamide-β-D-galactoside was present, only colonies of S. aureuswere observed (FIG. 22 d).

What is claimed is:
 1. A method of selectively inhibiting the growth of non-target bacteria cells in a mixed population of target and non-target bacteria cells, the method comprising the steps of: (a) contacting the mixed population with a growth medium containing a selective agent comprising a carrier moiety linked to a toxic moiety having a negative LogP; wherein the carrier moiety is linked to a toxic moiety; wherein the selective agent is able to enter non-target cells in which the linkage is cleaved, releasing the toxic moiety to exert a toxic effect on the non-target cells causing inhibition of the growth of the non-target cells, whereas the linkage is not cleaved in target cells or the toxic moiety, if released from the selective agent, does not exert a toxic effect on the target cell; and (b) culturing the bacteria cells in conditions which allow for growth of target cells,
 2. The method of claim 1, wherein the method is performed without contacting the mixed population with a pre-enrichment medium that lacks the selective agent.
 3. The method of claim 1, wherein, prior to the culturing step, the number of non-target cells in the mixed population is greater than the number of target cells in the mixed population.
 4. The method of claim 1, wherein the mixed population of cells is collected from a food sample.
 5. The method of claim 1, wherein the mixed population of cells is in a stressed state.
 6. The method of claim 5, wherein the stressed state is a heat stressed state.
 7. The method of claim 1, wherein the non-target cells comprise E. coli, E. faecalis, and/or other coliform bacteria.
 8. The method of claim 1, wherein the target cells comprise S. aureus.
 9. The method of claim 1, wherein the toxic moiety comprises metronidazole or a salt thereof.
 10. The method of claim 1, wherein the toxic moiety comprises sulfamethoxazole or a salt thereof.
 11. The method of claim 1, wherein the toxic moiety comprises aminoxy-L-alanine of a salt thereof.
 12. The method of claim 1, wherein the toxic moiety comprises hydrazino-L-alanine or a salt thereof.
 13. The method of claim 1, wherein the toxic moiety comprises β-chloro-L-alanine or a salt thereof.
 14. The method of claim 1, wherein the carrier moiety comprises a tryptophan residue moiety and/or a sarcosyl-alanine residue moiety, and the toxic moiety comprises aminoethylphosphonic acid (AEP) or a salt thereof.
 15. The method of claim 14, wherein the target cells comprise Salmonella spp.
 16. The method of claim 1, wherein the carrier moiety comprises an isoleucine residue moiety and/or a sarcosyl-alanine residue moiety, and the toxic moiety comprises aminoethylphosphonic acid (AEP) or a salt thereof.
 17. The method of claim 14, wherein the non-target cells comprise E. faecalis.
 18. The method 1 wherein the carrier moiety comprises β-D-galactoside and the toxic moiety comprises a sulfacetamide residue moiety.
 19. The method of claim 18 wherein the target cells comprise S. aureus and the non-target cells comprise E. faecalis. 